The invention relates generally to methods for determining the presence of active periodontal disease in mammals and specifically to those methods for determining the presence of periodontal disease which involve assaying crevicular fluid for the presence of elevated levels of aspartate aminotransferase. More specifically, the invention relates to improvements in such methods which provide for determination of the type and severity of periodontal disease.
Periodontal diseases are inflammatory diseases of microbial etiology affecting the supporting tissues of the teeth. The term "periodontal disease" encompasses two major and distinct subclasses of disease, gingivitis and periodontitis. Gingivitis is characterized by inflammation of the gums without bone loss or loss of connective tissue attachment. Gingivitis is a precondition for, but does not necessarily lead to, periodontitis which is characterized by progressive formation of periodontal pockets between the gum tissue and tooth, resulting from loss of connective tissue attachment and bone loss, eventually leading to tooth loss. Presently available methods of measuring periodontal disease include subjective observational indices such as those of Loe, H. and P. Silness, Acta Odont. Scand. 21:533 (1963) for gingivitis and Ramfjord, S., J. Periodontal. 30:602 (1967) for periodontitis. These indices for periodontitis are based on criteria such as bleeding on gentle probing, pocket depth, attachment loss, and radiographic evidence of bone loss. Unfortunately, these clinical indicators, with the exception of bleeding on probing, are generally acknowledged to be reflective of past disease and prior damage. Of these indicators, only bleeding on probing (bleeding of gum tissue due to probing of the gum line or pocket with a hard instrument, e.g. probe or curet) has been claimed to correlate with active periodontal disease. Nevertheless, bleeding itself is a subjective indicator of disease and the diagnostic value of bleeding on probing has been questioned as such bleeding appears to be associated with a high proportion of false positive indications of periodontal disease. See Haffajee, A.D., S. S. Socransky, and J. M. Goodson, J. Clin. Perio. 10:257-265 (1983).
Other methods have been proposed for the diagnosis of periodontal disease. Because both gingivitis and periodontitis are characterized by accumulation and flow of crevicular fluid (a transudate of serum) at the gingival sulcus and pockets, measurement of the volume of crevicular fluid present at a site has been proposed as a diagnostic for periodontal disease. An instrument known as the Periotron (Harco Electronics Ltd.; Winnipeg, Canada) makes use of this principle by galvanometrically measuring the volume of crevicular fluid absorbed by small strips of porous material known as Periopaper (Harco; Tustin, California) which are inserted into the crevicular space between the tooth and gum.
Still other methods relate to analysis of components of crevicular fluid for the diagnosis of periodontal disease. Kornman, J. Period. Res., 22, (1987) discloses methods correlating the presence of collagenase in crevicular fluid with the severity of periodontal disease. A device (Periocheck, Advanced Clinical Technologies, Inc., Westwood, Mass.) is available which assays for neutral proteases to determine the presence of periodontal disease. The source of both collagenase and neutral proteases has been suggested to be polymorphonuclear leucocytes (PMN) migrating into the crevice. Other components of crevicular fluid such as chondritin-4-sulfate, the presence of which is considered indicative of bone destruction, have been found to differ in crevicular fluid associated with gingivitis and crevicular fluid associated with periodontitis. Prostaglandin E.sub.2, a mediator of inflammation, has also been indicated to be associated more closely with periodontitis than with gingivitis.
Of particular interest to the present invention is the disclosure that the presence of elevated levels of the enzyme aspartate aminotransferase (AST) in crevicular fluid is highly correlative of the presence of active periodontal disease. The correlation was first related in two abstracts presented at the American Association for Dental Research Meetings, Cincinnati, Ohio, Mar. 17-20, 1983 (Crawford, J.M., S. Mukherjee, D.A. Chambers, and R. Cohen, Abstract No. 241, and Mukherjee, S., J. Crawford, D.A. Chambers, and R Cohen, Abstract No. 242 both published on or after February 1, 1983) and in an article, Chambers, D.A., J.M. Crawford, S. Mukherjee and R. Cohen, J. Periodon., 55, No. 9, 526-530 (September, 1984). The Crawford et al., abstract discloses a study with dogs in which gingivitis and periodontitis were induced experimentally. Specifically, gingival health was established in five beagles and gingivitis was then allowed to develop for four weeks by introduction of a soft diet and withdrawal of brushing. Periodontitis was then induced by ligation of the dogs' teeth. Crevicular fluid samples were collected at weekly intervals in volumetric capillary tubes after isolation and drying of teeth. The abstract indicates that crevicular fluid obtained during the incidence of experimental periodontitis contained concentrations of AST (3209.+-.1435 SFU/ml) approximately ten-fold higher at their peak than prior to ligation (468.+-.164 SFU/ml) and further that crevicular fluid during the incidence of experimental gingivitis contained approximately ten-fold higher concentrations of AST than in serum (41.+-.4 SFU/ml).
The Chambers et al., J. Periodontal. article describes the dog study in greater detail and notes that the average AST levels in crevicular fluid correlated neither with clinical estimations of attachment levels nor with gingival inflammation. The article did note, however, that the AST peak occurring 2 weeks after ligation of teeth did coincide with the period of high levels of soft tissue destruction and osteoclast activity reported in the beagle dog model and with the period of active bone resorption in ligature-induced periodontitis in the monkey model. The article also disclosed that the level of AST in crevicular fluid did not correlate with enzyme levels in dental plaque, suggesting the absence of bacterial origin for the enzyme.
The Mukherjee Abstract describes measurement of AST levels in human crevicular fluid collected in volumetric capillary tubes from areas diagnosed as having gingivitis or periodontitis according to the periodontal disease index (PDI) of Ramfjord. Disease activity indicated by the presence or absence of bleeding upon probing was also noted. AST concentrations of crevicular fluid collected from areas showing no bleeding on probing=0 SFU/ml (N=4), minimal bleeding=464.+-.113 SFU/ml (N=4) and definite bleeding 595.+-.192 SFU/ml (N=6). The analysis of data sorted according to gingivitis and periodontitis showed 363.+-.182 SFU/ml (N=4) and 424.+-.119 SFU/ml (N=3) respectively. The Abstract notes that the level of AST in crevicular fluid may correlate with the disease activity as determined by bleeding on probing.
While the references fail to demonstrate a specific positive association between elevated AST activity in gingival crevicular fluid and either attachment loss or gingival inflammation, they do indicate that there exists a general association between elevated AST levels in gingival crevicular fluid and periodontal disease activity as determined by bleeding on probing. Chambers, European Patent Application No. 151,536 published August 14, 1985 based on U.S. Pat. application Ser. No. 575,552 filed Jan. 31, 1984, since abandoned and continued as U.S. Ser. No. 06/814,065 filed Dec. 19, 1985, since abandoned, and continued as U.S. Ser. No. 07/096,803 filed Sept. 10, 1987, since abandoned, and continued as U.S. Ser. No. 07/317,138 filed Mar. 1, 1989 the disclosure of which is hereby incorporated by reference, relates to the work embodied in the article and abstracts and the recognition of the general relationship between elevated AST levels and periodontal disease activity. The application claims diagnostic methods based on the recognition that the presence of elevated levels of AST in crevicular fluid is predictive of a high probability of progressive, as opposed to nonprogressive, periodontal disease and corresponding tissue damage.
According to the method of the Chambers patent application, crevicular fluid is collected from the gingival sulcus by means such as a microsyringe, capillary tube or absorbant strip. The volume of material is measured and the concentration of AST in the collected sample of crevicular fluid is determined by either colorimetric or immunological assay. The patent application claims a method for determining the presence of active periodontal disease in mammals comprising assaying crevicular fluid for the presence of elevated levels of aspartate aminotransferase. The application defines elevated levels as being an amount of AST substantially in excess of the level of AST normally found in the blood stream of healthy adults of the species being tested which ranges from about 4 to about 32 milli-International Units/ml (mIU/ml) depending upon the precise testing protocol used.
Since the original reports of work by the Chambers, et al. group on AST, the relationship between other tissue and bacterial enzymes and periodontal diseases has been studied. Lamster, et al., J. Periodontal., 56, 139-147 (1985), disclose studies assessing crevicular fluid volume and activity of the enzymes lactate dehydrogenase (LDH), .beta.-glucuronidase (BG) and arylsulfatase (AS) in crevicular fluid during the development of experimental gingivitis.
LDH is said to be derived primarily from cells in the sulcular epithelium, but that fibroblasts and polymorphonuclear leucocytes (PMN) that lyse in the crevice also contribute to the LDH pool. BG is said to be primarily derived from degradation of lysosomal granules of PMN with macrophates contributing to the BG pool. The pattern of AS activity was characterized as being between that of LDH and BG with sources of this enzyme including PMN, mast cells and fibroblasts.
Crevicular fluid "resting" volumes were determined by inserting a filter paper strip into the gingival sulcus until mild resistance was felt and leaving the strip in place for 30 seconds before determining the volume of fluid absorbed. After removal of the strip, the crevicular fluid "flow" volume was determined by waiting 30 seconds and inserting a second filter paper strip into the site for 3 seconds. Analysis of data collected from subjects subjected to experimental gingivitis indicated that while clinical inflammation increased during the 4 weeks of the study, the concentrations and total activity (concentration times sample volume) of BG and AS rose during the onset of gingivitis but peaked or leveled off after reaching a maximum 2 or 3 weeks into the study. The data indicated that an increase in fluid volume without a corresponding increase in BG or AS activity occurred during the latter part of the trial. The increase in LDH concentration and total activity during the experiment was not dramatic and was consistent with an earlier experiment wherein the concentration of LDH in crevicular fluid was higher in subjects with healthy gingiva than with mildly inflamed gingiva. Lamster, et al. also suggested that reporting of crevicular fluid constituent data in terms of concentration alone might be inadequate and that it might be desirable to report enzyme data in terms of both concentration and total activity of the sample.
Lamster, et al., J. Clin. Periodontol., 13, 799-804 (1986) present data wherein LDH, BG and AS concentrations and total activities for 30 second samples were assayed for a group of periodontitis patients and a control group. Negative or low positive correlation coefficients were determined between enzyme concentrations and gingival index (GI) and probing depth. On the other hand, "a modest, but not absolute" correlation between increasing severity of pathology and total enzyme activity for a 30 second sample was suggested by the data. Lamster, et al. suggested therefore that total activity in a standardized sample might be a more appropriate means of reporting crevicular fluid constituent data.
In spite of the various advances made in the art, the art still fails to provide a simple, reliable means for an enzymatic determination of the type and severity of periodontal disease. Such a method might be used for diagnosis of such disease or for determination of the efficacy of treatment of that periodontal disease condition. There currently exists no completely suitable means for determining the efficacy of treatment for periodontal disease. Such monitoring is a nontrivial concern because of the serious nature of continuing or repeating treatments for periodontitis involving administration of drugs, root planing or surgery. Current methods comprise monitoring of the clinical parameters of gingival bleeding, probing depths and radiographic interpretations of the bone. Because monitoring of probing depths and bone levels provides only a belated evaluation of treatment efficacy, an improved approach is clearly desired.